Until last year, the humoral control of megalkaryocyte and platelet production remained enigmatic. In 1994, several groups, including our own cloned Thrombopoietin (TPO), the crucial regulator of thrombopoiesis. Its biological characteriztion will surely advance our understanding of blood cell development, and its clinical use will very likely enhance the care of patients suffering from marrow failure states. However, little is presently known about the structural features of the protein which account for its biological activities. We propose to test a model of thrombopoietin structure and function. We will define regions of TPO responsible for structural integrity, receptor binding and functional activity by engineering murine TPO mutants using site-directed mutagenesis and homologous substitution, and test the resulting muteins for binding and function. The strong homology between erythropoietin (EPO) and TPO, and computer based modeling strategies will allow focused targeting of mutagenesis sites to regions known to be critical for the function of EPO and for other members of the cytokine family. Epitope mapping and critical regions identified by homologous substitution will allow narrowed targeting of mutagenesis, refinement of the model and ultimately identification of the individual residues critical for receptor docking and signaling. The ensuing knowledge of structure and function will advance our understanding of the structural basis for protein-protein interactions, and may provide a foundation for rational drug design.